Unlike adaptive lymphocytes, innate and innate-like lymphocytes acquire stereotypic polarized helper effector programs during development, independently of pathogen exposure and prior to taking up residence in peripheral tissues where they function as first line of defense against aggressions by pathogens, allergens, tumors and injuries. Our group has established that PLZF encoded by Zbtb16 is a signature master transcription factor directing the acquisition of innate effector programs across many of these innate lineages, including CD1d-restricted NKT cells, MR1-restricted MAIT cells, ?? T cell subsets and innate lymphoid cells (ILC). While the genetic mechanisms of activation of this effector program by PLZF is now well dissected, this project specifically focuses on the conditions leading to the expression of PLZF during lymphoid development. Since PLZF is necessary and sufficient for activation of the innate effector program, the central question of this proposal revolves around the regulation of PLZF expression itself. Our general hypothesis is that specific transcriptional regulatory networks act upon distinct or partially overlapping sets of enhancers to govern Zbtb16 expression in different innate lineages. We will use recent technical advances that allow broad mapping of genomic regions of chromatin accessibility (ATAC-seq) and their systematic deletions (Crispr-Cas9) to comprehensively dissect the enhancer landscape (regulome) of Zbtb16 in vivo (Specific Aim 1). Through advanced computational motif analysis, we will then identify candidate transcription factors (trans-regulation) and evaluate their contributions to the regulation of Zbtb16 expression (Specific Aim 2). Finally, we will examine the mutual antagonism between PLZF and Bach2, to understand how they reciprocally regulate the innate and adaptive pathways of lymphocyte development (Specific Aim 3). Collectively, these specific aims address the fundamental dichotomy between innate and adaptive lymphocyte development and will generate knowledge and tools to selectively manipulate their properties for therapeutic benefit in health and disease.